Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/585—Calcitonins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to novel polypeptides of the formula ##STR2## wherein A 1 is Ser or Gly, A 7 is Val or Met, A 10 is Lys or Thr, A 11 is Leu or Tyr, A 12 is Ser or Thr, A 14 is Glu or Asp, A 15 is Leu or Phe, A 16 is His or Asn, A 18 is Leu or Phe, A 19 is Gln or His, A 21 is Tyr or Phe, A 23 is Arg or Gln, A 25 is Asp or Asn or Ala, A 26 is Val or Thr or Ile, A 28 is Ala or Ser or Val, and A 30 is Thr or Val or Ala, and acid addition salts and complexes thereof.
• polypeptides of formula [I] have serum calcium reducing activity.
• Calcitronin is a well-known polypeptide having mammalian serum calcium reducing activity, and is isolated from mammalian thyroid gland or avian or piscine ultimo branchial body.
• the amino acid sequence of the calcitonin depends on the origin of the species and synthetic calcitonins having the same chemical structure as those of natural origin have recently become known.
• calcitonins of natural origin such as eel-, salmon- or human-calcitonin are polypeptides consisting of 32 amino acids, wherein the first and 7th amino acids are L-cysteine and the mercapto group thereof is bonded to comprise a disulfide bridge.
• polypeptide of the present invention has neither first nor seventh amino acid L-cysteine and the said L-cysteine is replaced by alpha-aminosuberic acid of the formula; ##STR3##
• the carboxyl group at ⁇ -position is bonded with N-terminal amino acid to form a ring structure.
• novel compounds have important therapeutic properties, and may be prescribed for the purpose of reducing serum calcium content in diseases such as hypercalcemia, an endogenous calcitonin deficiency disease caused by dysthyroidism or hyperparathyroidism.
• the novel compound can be prescribed for osteopathy requiring calcium, such as osteoporosis, ostermalacia, fracture, fibrous dysplasia of the bone or rachitis caused by corticosterone therapy or inactivation after menopause or external injury, and is especially suited to therapy in combination with calcium or phosphorus.
• the novel product can be used for therapy of Paget's disease and for therapy or prevention of peptic ulcer.
• the compound of the present invention is more stable in serum, liver or kidney than the known calcitonins, and is more stable in the purification process or storage, due to lack of disulfide linkage.
• the activity of some of the polypeptides of the formula [I] is the same or higher as compared with the known calcitonin on the tests upon rate.
• Synthesis of the compound [I] can be by the conventional peptide synthesis method, that is to say, an amino acid and/or peptide consisting of 2 - 4 amino acids is reacted by condensation in the order of the amino acid sequence of formula [I], and the amino acid construction unit which contains the sequence of the formula ##STR4## is subjected to ring formation at any stage of construction of the peptide unit, and the protective group for the reactive group is released at any stage of the reaction. If desired the product may be converted to its acid addition salt or complex.
• the protective groups for the synthesis of the starting materials or intermediates are conventional protective groups for peptide synthesis and should be easily removable by hydrolysis, acid decomposition, reduction, aminolysis or hydrazinolysis.
• the amino group may be protected conventionally by an acyl group such as formyl, trifluoroacetyl, phthaloyl, benzensulfonyl, p-toluenesulfonyl, o-nitrophenylsulfenyl or 2,4-dinitrophenylsulfenyl group; an aralkyl group such as benzyl, diphenylmethyl or triphenylmethyl (these groups may optionally substituted with a lower alkoxy group such as o-methoxy or p-methoxy); a benzyloxycarbonyl group such as benzyloxycarbonyl, o-bromobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, o-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-phenylazo-benzyloxycarbonyl group
• the carboxyl group can be protected by its amide formation, hydrazide formation or esterification.
• the amide group is substituted with a 3,4-dimethoxybenzyl or bis-(p-methoxyphenyl)-methyl group.
• the hydrazide group is substituted with a benzyloxycarbonyl, trichloroethyloxycarbonyl, trifluoroacetyl, t-butoxycarbonyl, trityl or 2-p-diphenyl-isopropoxycarbonyl group.
• the ester group is substituted with an alkanol such as methanol, ethanol, t-butanol or cyanomethylalcohol; an aralkanol such as benzylalcohol, p-bromobenzylalcohol, p-chlorobenzylalcohol, p-methoxybenzylalcohol, p-nitrobenzylalcohol, 2,4,6-trimethylbenzylalcohol, benzhydrylalcohol, benzoylmethylalcohol, p-bromobenzoylmethylalcohol or p-chlorobenzoylmethylalcohol; a phenol such as 2,4,6-trichlorophenol, pentachlorophenol, p-nitrophenol, 2,4-dinitrophenol, p-cyanophenol or p-methanesulfonylphenol; or a thiophenol such as thiophenol, thiocresol or p-nitrothiophenol.
• the hydroxy group in serine, threonine or tyrosine may optionally be protected by esterification or etherification.
• a group protected by esterification is, for example, a lower alkanoyl group such as an acetyl group; an aroyl group such as a benzoyl group; or a group derived from carbonyl such as benzyloxycarbonyl or ethyloxycarbonyl.
• a group protected by etherification is, for example, a benzyl, tetrahydropyranyl or t-butyl group.
• Protection of the hydroxy group can be effected by a 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl or 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group. However it is not always necessary to protect these hydroxy groups.
• the amino group or guanidino group in arginine can be protected by a nitro, tosyl or benzyl oxycarbonyl group, however the guanidino group does not always require protection.
• the imino group in histidine can be protected by a benzyl, trityl, benzyloxycarbonyl, tosyl, admantyloxycarbonyl, 2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl or 2,2,2-trifluoro-1-benzyloxycarbonylaminoethyl group, although the imino group does not always require to be protected.
• the peptides of the starting materials or intermediates are synthesized by condensation of amino acids or peptides--preferably of two to four amino acids--in the order of the amino acid sequence of formula [I]. For example, an amino acid or peptide having a protected ⁇ -amino group and activated terminal carboxyl group is reacted with an amino acid or peptide having free ⁇ -amino group and protected terminal carboxyl group. On the other hand, an amino acid or peptide having activated ⁇ -amino group and protected terminal carboxyl group is reacted with amino acid or peptide having free terminal carboxyl group and protected ⁇ -amino group.
• the carboxyl group can be activated by, for example, an acid azide, acid anhydride, acid imidazolide or active ester, such as by converting to cyanomethyl ester, thiophenylester, p-nitrophenylester, p-nitrothiophenylester, p-methanesulfonylphenylester, thiodylester, 2,4-dinitrophenylester, 2,4,5-trichlorophenylester, 2,4,6-trichlorophenylester, pentachlorophenylester, N-hydroxyphthalimidoester, 8-hydroxypiperidine ester or N-hydroxypiperidine ester, carbodiimide, N,N'-carbonyl-diimidazol or an isoxazolium salt such as Woodward reagent.
• active ester such as by converting to cyanomethyl ester, thiophenylester, p-nitrophenylester, p-nitrothiopheny
• the carboxyl group can be activated in a conventional way by, for example, an acid azide, acid anhydride, active ester or carbodiimide.
• condensation reactions are the Wunsch method, azide, active ester or Geiger methods. In the condensation reaction, racemization should carefully be avoided.
• the ring formation reaction is performed by a condensation reaction with activated ⁇ -carboxyl group in ⁇ -amino suberic acid and free amino group in N-terminal amino acid.
• the condensation reaction the hydroxyl group of serine and threonine is preferably protected.
• the preferred process of the present invention is preceded by a condensation reaction with a peptide ring (having optionally protected active groups) containing ⁇ -amino suberic acid and the remaining other large peptide having optionally protected active groups. That is to say, the N-terminal fragment consisting of the first to sixth - ninth amino acid is condensed with the residual peptide of the total sequence of seventh - tenth to thirty-first amino acids. To promote reactivity of both fragments at condensation and to prevent racemization, glycine is preferably used as the C-terminal amino acid.
• a peptide consisting of first - ninth amino acid sequence is reacted with a peptide consisting of 10th - 31st amino acid sequence.
• the said condensation reaction can be achieved by starting from the peptide having free terminal carboxyl group, the so-called Wunsch method, or by analogous methods. Also it can preferably be achieved by the azide method starting from the peptide having an azide or hydrazide terminal, by the active ester method, or by the mixed anhydride method.
• N-terminal fragment such as nonapeptide consisting of first to ninth amino acid sequence will be explained in detail in the following, however hexapeptide 1-6, heptapeptide 1-7, or octapeptide 1-8 can also be produced by substantially the same process.
• the nonapeptide sequence can be produced by connecting each amino acid with lower peptide consisting of 2-4 amino acids in the order of amino acid sequence from C-terminal amino acid.
• An amino acid such as glycine, leucine, valine, ⁇ -amino suberic acid, threonine, methionine, serine or asparagine is preferably condensed by the active ester method.
• the lower peptide such as tripeptide 2-4 is preferably condensed by the Geiger method or by the Wunsch method.
• the carboxyl group can also be protected by esterification by alcohols such as methanol, benzyl alcohol or other alcohols.
• the ester group such as methyl ester can be removed by dilute sodium hydroxide solution or by conversion to hydrazide, and the benzyl ester group can be removed by catalytic hydrogenation.
• the amino group of the intermediate is protected by a conventional protective group, such as a benzyloxycarbonyl, trityl, t-butoxycarbonyl or 2-p-diphenyl-isopropoxycarbonyl group.
• the carboxyl group can be protected, if required, by conventional esterification.
• the hydroxyl group in serine and threonine can be protected, if necessary, by esterification using t-butanol, benzyl alcohol or other alcohol.
• the benzyloxycarbonyl, p-nitrobenzylester and benzyl ester groups are split by catalytic hydrogenation in the presence of palladium carbon.
• the N-trityl group is split by aqueous acetic acid, and the t-butoxycarbonyl group can be decomposed by trifluoroacetic acid.
• the o-nitrophenylsulfenyl group is split by hydrogen chloride, hydrogen cyanide or sulfurous acid in organic solvent.
• the diphenylisopropoxycarbonyl group is split by a mixture of acetic acid-formic acid-water (7:1:2).
• Methyl ester, ethyl ester or p-nitrobenzyl ester is changed to hydrazide by using hydrazine hydrate.
• the methyl ester group can be split by dilute sodium hydroxide solution, and t-butyl ester is split by trifluoroacetic acid.
• a peptide having C-terminal consisting of amino acid sequence from seventh - tenth to thirty-first, which is condensed with N-terminal peptide hereinabove, is preferably synthesized by connecting C-terminal amino acid (amino acid No. 31) or C-terminal fragment, such as peptide of amino acid sequence 30th-31st, 28th-31st, 25th-31st, 24th-31st or 23rd-31st, with each amino acid or lower peptide consisting of two to four amino acids in the order of foregoing amino acid sequence.
• C-terminal fragment 10th - 31st can be produced by condensation of amino acids and lower peptides such as dipeptide 28th-29th, dipeptide 26th-27th, tripeptide 20th-22nd, tetrapeptide 15th-18th and tripeptide 10th-12th in the order of amino acid sequence from the C-terminal, by the active ester method or the Geiger method.
• Preferred protective groups for each group are: ⁇ -amino group by t-butoxycarbonyl group; side chain carboxyl group of aspartic acid and glutamic acid by benzyl ester group; ⁇ -amino group of lysine by o-chlorobenzyloxycarbonyl group; hydroxyl group of serine, threonine and tyrosine by benzyl group; and amino group in the guanidine group of arginine and imino group of histidine by tosyl group.
• the protective group of C-terminal fragment seventh-10th to 31st having protected ⁇ -amino group hereinabove, for example docosapeptide amide of 10th-31st sequence, is removed by a suitable method.
• the trityl group is split by aqueous acetic acid.
• Diphenyl isopropoxycarbonyl, benzyloxycarbonyl and t-butoxycarbonyl groups are removed by a mixture of glacial acetic acid, formic acid and water; hydrogenation; and trifluoroacetic acid, respectively.
• the hentriacontapeptideamide having protected ⁇ -amino group, protected ⁇ -amino group, optionally protected side chain carboxyl group and/or hydroxyl group is obtained.
• These protective groups are split by the process hereinbefore described preferably by acid decomposition such as by hydrogen fluoride, and finally the product [I] can be obtained.
• the hydroxy group in serine, threonine and tyrosine can be protected by, for example, benzyl; the imino group in histidine can be protected, for example, by 1-benzyloxycarbonylamino-2,2,2-trifluoroethyl group; the guanidino group in arginine can be protected, for example, by a nitro group; and the side chain in glutamic acid can be protected, for example by a benzyl ester group.
• the protective group for the ⁇ -amino group is, for example, a t-butyloxycarbonyl, o-chlorobenzyloxycarbonyl or o-bromobenzyloxycarbonyl group.
• the protected peptide is removed from the carrier resin and the protective group is removed by anhydrous hydrogen fluoride.
• One-step removal of all protective groups by hydrolysis using trifluoroacetic acid can be achieved when t-butoxycarbonyl group is used for amino group protection; t-butyl ester for side-chain carboxyl group protection; t-butyl ether for hydroxyl group protection in serine; and threonine, tyrosine and 2,2,2-trifluoro-1-t-butoxycarbonylaminoethyl group for imino group protection in histidine.
• the novel polypeptide [I] of the present invention can be obtained in the form of a free base or a salt thereof.
• the free base may conventionally be obtained from its salt.
• the free base can be changed to its pharmacologically acceptable salt by reacting with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid, or an organic acid such as formic acid, acetic acid, propionic acid, glycole acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, citric acid, tartaric acid, benzoic acid, benzenesulfonic acid or toluenesulfonic acid.
• an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, or phosphoric acid
• an organic acid such as formic acid, acetic acid, propionic acid, glycole acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, mal
• the novel polypeptide can be converted into a complex thereof by addition of various inorganic or organic substances.
• the said complex form is composed by adding a kind of inorganic or organic substance to a long chain polypeptide, the complex having a long term activity when administered.
• the said complex-forming substance are inorganic compounds derived from metals such as calcium, magnesium or zinc, especially phosphate, pyrophosphate or polyphosphate of the said metals.
• Further examples of the said complex-forming organic substance are non-antigenic gelatine, CMC, polyglutamic acid or the like.
• the assay method for serum calcium reducing activity, the carrier and developing system for thin layer chromatography and conditions for amino acid analysis are as follows:
• Sample is diluted adequately with 0.1 N sodium acetate - 0.1% albumin solution.
• Male rats are subjected individually to intravenous injection with 0.2 ml. of the respective diluted solutions. After 1 hour, all the rats are killed to obtain their respective blood, and serum calcium value of each blood sample is determined by atomic absorption spectrophotomery.
• Research Standard B which is an extract obtained from the thyroid gland of the hog, is diluted so as to give dilutions of 2.5, 5, 10 and 20 MRC mU/0.2 ml. respectively. Male rats are subjected to intravenous injection with 0.2 ml. of the respective diluted solutions. Following the same procedure as above, the serum calcium value of the rats is determined one hour after injection. From the potency of the corresponding Research Standard, the potency of the calcitonin in the sample is determined.
• Carrier silica gel G.
• Sample is hydrolyzed with 6 N HCl (with addition of a few drops of anisole) at 110° C., for 40 - 45 hours, and dried out under reduced pressure, then is subjected to amino acid analysis.
• the starting material is produced as follows:
• BOC-Thr(Bzl)-Pro-NH 2 (10.0 g.) was added to TFA (30 ml.) at -5° C., stirred for 30 minutes, and concentrated in vacuo. The residue was treated with ether, filtered and dried over sodium to obtain H-Thr(Bzl)-Pro-NH 2 .TFA.
• BOC-Ala-Gly-OH obtained in (3) (6.08 g.) and HOBT (3.34 g.) were added in THF (100 ml.) and WSC (4.5 ml.) was added thereto at -5° C. (pH 4.0). After stirring at -5° C.
• BOC-Val-OH.DCHA (219.2 g.) was added to ethyl acetate (1 liter). The solution was washed twice with 1 N HCl (600 ml.) and water (500 ml.) in this order. After drying with anhydrous sodium sulfate, the solution was concentrated in vacuo. THF (100 ml.) and dichloromethane (400 ml.) were added to the residue and H-Gly-OEt.HCl (69.8 g.) and triethylamine (70 ml.) added thereto. DCC (103.2 g.) was added thereto at -5° C. and stirred at -5° C. for 1 hour and at room temperature overnight.
• BOC-Val-Gly-OEt (96.8 g.) was dissolved in methanol (150 ml.) and 1 N NaOH solution (368 ml.) at -5° C. was added thereto. After 1 hour of stirring, the pH was adjusted to 8.0 by addition of 1 N HCl. Methanol was removed by concentration in vacuo and the aqueous layer was washed with ethyl ether (100 ml.). The aqueous layer was adjusted to pH 2.0 by addition of 1 N HCl. The aqueous layer was extracted four times with ethyl acetate (200 ml.), and the extract was dried using anhydrous sodium sulfate and thereafter concentrated in vacuo.
• BOC-Ala-Gly-Thr(Bzl)-Pro-NH 2 (10.5 g.) was added to TFA (35 ml.) at -5° C., and, after stirring for 30 minutes, concentrated in vacuo. The residue was treated with ethyl ether, and the precipitated material was filtered and dried in vacuo over sodium hydroxide to obtain H-Ala-Gly-Thr(Bzl)-Pro-NH 2 .TFA. THF (100 ml.) was added thereto, and triethylamine was added at -5° C. to adjust the pH to 5.0. BOC-Val-Gly-OH (5.49 g.) and HOBT (2.66 g.) were added thereto.
• the pH was adjusted during stirring, to pH 7.5, by adding N-methylmorpholine, then adding BOC-Asp(OBzl)-OSU (2.0 g.) again, adjusting the pH to 7 by addition of N-methylmorpholine, and the mixture was stirred overnight.
• a large amount of water was added to the reaction mixture, and the precipitated sticky substance was separated by decantation to crystallize by treatment with ethyl ether.
• the aqueous layer was extracted with chloroform and the extract was concentrated in vacuo. The residue was treated by addition of water to precipitate a sticky substance.
• the precipitated sticky substance was crystallized by treatment with ethyl ether.
• TFA (20 ml.) was added at -5° C. to BOC-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH 2 (6.52 g.); stirred for 30 minutes and concentrated in vacuo. The residue was treated by adding ethyl ether and the precipitated substance was dried in vacuo over NaOH. DMF (25 ml.) was added thereto and the pH was adjusted to 5.0 by adding triethylamine at -5° C.
• Amino acid composition NH 3 1.28, Arg 0.95 (1), Asp 1.04 (1), Thr 1.50 (2), Pro 0.98 (1), Gly 1.84 (2), Ala 1.00 (1), Val 1.01 (1).
• BOC-Tyr(Bzl)-OH.CHA (37.6 g.) was mixed with ethyl acetate (300 ml.) and 1 N HCl (120 ml.), separated and the ethyl acetate layer washed three times with water. After drying with anhydrous sodium sulfate, the organic layer was concentrated in vacuo and the oily residue was dissolved in dichloromethane (80 ml.). H-Pro-Obzl.HCl (19.3 g.) was then added thereto and WSC (14.6 ml.) was added to the mixture at -5° C, then the mixture was stirred for 1 hour at -5° C. and overnight at room temperature.
• the reaction mixture was concentrated in vacuo, and the residue was shaken with ethyl acetate (800 ml.) and 1 N HCl (400 ml.) to separate the ethyl acetate layer.
• the organic layer was washed twice with 1 N HCl (300 ml.), twice with water (300 ml.), three times with 5% aqueous sodium bicarbonate (300 ml.) and twice with water (300 ml.), in this order.
• the organic layer was dried with sodium carbonate and concentrated in vacuo.
• the crude product was subjected to column chromatography using silica gel (400 g.).
• AOC-Arg(Tos)-Thr(Bzl)-Asp(OBzl)-Val-Gly-Ala-Gly-Thr(Bzl)-Pro-NH 2 (6.0 g.) was added in TFA (20 ml.) at -5° C., stirred for 30 minutes and concentrated under reduced pressure. The residue was treated with ethyl ether and dried in vacuo over NaOH.
• Amino acid analysis NH 3 1.60, Arg 0.92 (1), Asp 1.04 (1), Thr 2.1 (3), Pro 2.08 (2), Gly 1.86 (2), Val 1.00 (1), Tyr 0.91 (1).
• H-His-OH.HCl (3.83 g.) was dissolved with heating in water (25 ml.). After cooling to room temperature, sodium bicarbonate (1.84 g.) was added, and, further, HOBT (270 mg.), BOC-Leu-OSU (8.53 g.) and THF (25 ml.) were added. Water (10 ml.) was then added and the mixture stirred overnight at room temperature. The pH of the reaction mixture was adjusted to 7 with addition of aqueous sodium bicarbonate, and BOC-Leu-OSU (1.3 g.) was again added and the mixture stirred. After 6 hours, a further 1.3 g. was added and stirring continued overnight.
• the reaction mixture was concentrated in vacuo to remove organic solvent and the residual aqueous layer was washed with ethyl acetate.
• the aqueous layer was adjusted to pH 5 and poured into the top of the column of HP-20 (2.3 ⁇ 18 cm.), followed by washing with water until the washing eluate showed ninhydrin negative, then eluted with methanol.
• the eluate was concentrated in vacuo and the residue was treated with ethyl ether.
• the precipitate formed was recovered by filtration and reprecipitated twice with methanol - ethyl ether to obtain dried powder (3.94 g., Yield: 53.5%, m.p.: 178° - 180° C.).
• H-Ser(Bzl)-OH (11.7 g.) was dissolved in water (50 ml.) and sodium bicarbonate (10.1 g.) and dioxane (10 ml.) added thereto.
• BOC-Leu-OSU (16.4 g.) in dioxane (15 ml.) and DMF (5 ml.) were then added.
• the pH of the reaction mixture was adjusted to pH 7, and the mixture concentrated in vacuo to remove organic solvent.
• the aqueous layer was shaken with ethyl acetate (100 ml.) and the organic layer was washed twice with 1 N HCl and water.
• the crystals were dissolved in DMF (8 ml.) and BOC-Lys(ClCbz)-ONP (8 g.) and HOBT (270 mg.) were added thereto.
• the pH of the solution was adjusted to pH 8 with N-methylmorpholine then stirred overnight at room temperature.
• 1 N HCl was added thereto and the precipitate was dissolved in chloroform which was washed three times with 1 HCl, three times with 5% sodium bicarbonate solution and three times with water. After drying with anhydrous magnesium sulfate, the chloroform layer was concentrated in vacuo.
• the substance hereinabove was dissolved in DMF (15 ml.), the pH adjusted to 5.5 to 6 by adding N-methylmorpholine, HOBT (360 mg.), BOC-Lys(ClCbz)-Leu-Ser(Bzl)-OH (1.86 g.) and DCC (540 mg.) dissolved in DMF (5 ml.) added, then the mixture was stirred for 1 hour and overnight at room temperature. 1 N HCl was added and the precipitate formed was collected by filtration. The ethanol-insoluble part of the precipitate and the precipitated material obtained by addition of ethyl ether in the filtrate were combined.
• Amino acid sequence No. 1 - 9 ##STR9## is produced as follows.
• N,N-dimethylamino-1,3-propanediamine was added, stirred for 3 hours, then concentrated up to 100 ml., which was extracted with ethyl acetate. The ethyl acetate layer was washed with 1 N HCl and water, and was distilled in vacuo. The thus-obtained oily product was dissolved in ether and transferred to 5% bicarbonate solution.
• the reaction mixture was adjusted to pH 6 by adding N-methylmorpholine and stirred for 3 days at room temperature. N,N-dimethylamino-1,3-propanediamine was added thereto and the mixture stirred for 1 hour, then water was added and extracted with ethyl acetate. The extract was washed with 1 N HCl, water, 5% sodium bicarbonate and water in this order and dried with anhydrous sodium sulfate. Ethyl acetate was removed and the residue was dissolved in ethyl ether (300 ml.), then re-extracted with 5% sodium bicarbonate solution.
• the aqueous layer was acidified with HCl, extracted with ethyl acetate and the ethyl acetate layer washed with 5% sodium bicarbonate solution and water, then dried with anhydrous sodium sulfate. After an equal amount of cyclohexylamine was added to the ethyl acetate layer, the mixture was distilled under reduced pressure to obtain oily product which was solidified by adding ether and n-hexane.
• BOC-Ser(Bzl)-Asn-Leu-NHNH 2 (8.5 g.) was dissolved in DMF (30 ml.), and dioxane (14 ml.) was added containing 1 N HCl and isoamyl nitrite (3.1 ml.), then reacted for 20 minutes to form the azide.
• the dried TFA salt obtained hereinbefore was dissolved in DMF (10 ml.), neutralized with triethylamine, and then slowly added to the solution containing the azide compound at below -40° C.
• the pH was adjusted to 7 by adding triethylamine and reacted for 3 days at 5° C.
• the lyophilizate was dissolved in 0.1 M AcOH and chromatographed through a column of Sephadez G-50 (2.2 ⁇ 105 cm.) by elution with 0.1 M AcOH (10 ml./hour). The eluate (5 g.) was fractionated, then the active fractions (Nos. 55- 63) were collected and lyophilized to obtain the active powder (22 mg.).
• the active powder (22 mg.) dissolved in 0.01 M aqueous ammonium acetate was charged on a column packed with CM-cellulose (2.3 ⁇ 25 cm.), then was gradiently eluted with 600 ml. of 0.01 mole to 600 ml. of 0.1 mole ammonium acetate (pH 4.5). Each 8 g. of eluted fraction was collected and the active fractions Nos. 109 - 119 were freeze dried.
• This powder dissolved in a small amount of 0.1 mole acetic acid was poured into the top of a column of Sephadez LH-20 (2.3 ⁇ 135 cm.), eluted with 0.1 mole acetic acid, fractions comprising each 6 g. of eluate were taken and the active fractions Nos. 31 - 34 collected which were freeze dried to obtain the active powder.

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